As one method for identifying a protein or peptide or for determining its amino acid sequence, a method employing mass spectrometry has been widely used.
A typical procedure of the analysis is as follows: Initially, an MSn analysis for a protein or peptide to be analyzed is performed using a mass spectrometer capable of an MSn analysis, such as an ion trap time-of-flight mass spectrometer. That is to say, ions generated from a target protein or peptide are dissociated by an appropriate dissociating technique to produce various fragment ions (product ions) which are fragments of the original ions. These fragment ions are separated according to their mass-to-charge ratios m/z and subsequently detected to create a mass spectrum of the fragment ions.
The most common technique for dissociating ions originating from a protein or peptide is the collision induced dissociation, in which ions are made to collide with gas (normally, inert gas) to induce dissociation. Other techniques, such as an electron transfer dissociation (ETD) or electron capture dissociation (ECD), are also frequently used for dissociating ions originating from a protein or peptide. The ETD and ECD are radical induced dissociation methods which use charged particles. A radical induced dissociation method in which neutral radical particles that are not electrically charged are used in place of the charged particles is also commonly known. An example of this type of radical induced dissociation method is an ion dissociation method disclosed in Patent Literature 1 in which ions are irradiated with hydroxy radicals (OH radicals).
The most common technique for identifying a protein or peptide using a mass spectrum obtained by a previously described kind of measurement is a database search (see Non Patent Literature 1). This technique uses a database which holds amino acid sequences and other items of information on known kinds of proteins or peptides. Mass data of the fragment-ion peaks appearing on a measured mass spectrum are compared with those of the fragments theoretically calculated from the amino acid sequence of each of the proteins or peptides contained in the database. Based on the degree of matching of the two sets of data, the most likely protein or peptide is chosen as the identification result, or a plurality of candidates which are likely to be the protein or peptide concerned are presented as the identification result. For example, an MS/MS ion search included in “Mascot”, which is an analysis software system for database search offered by Matrix Science Inc, can be used for such a database search. As for the database, various databases which are available to the public, e.g. Swiss-Prot, can be used.
There is also a method in which an amino acid sequence is estimated by finding an amino acid that matches the mass difference between the neighboring peaks appearing on a mass spectrum of fragment ions, instead of using a database. This is the technique called “de novo sequencing”. In this case, once the amino acid sequence is determined by de novo sequencing, the protein or peptide can be identified by searching a database for a protein or peptide that corresponds to that amino acid sequence.
In any of the previously described cases which employ database searches or de novo sequencing for identifying a protein or peptide, if there are too many kinds of fragment ions observed on the mass spectrum obtained by a measurement, it is difficult to assign the fragment ions, i.e. to estimate the kinds of fragments corresponding to those fragment ions. This may prevent successful identification of the protein or peptide or cause a significant deterioration in the identification accuracy. There are some possible causes of this problem.
For example, even when the origin of a fragment ion observed in a measurement is a known protein or peptide already recorded in a database, if the mechanism of the fragmentation in the protein or peptide concerned has not yet been fully clarified, the fragment ion may not be present in a list of theoretical fragments corresponding to that protein or peptide by an analysis software program. In such a case, the fragment ion observed in the measurement does not match any of the theoretical fragments. Consequently, the degree of matching may become so low that the protein or peptide cannot be identified.
Besides, the mass spectrum of fragment ions should preferably originate from a single protein or peptide. However, if the isolation of the target ion (selection of the precursor ion) before the dissociation of ions is insufficient, ions originating from two or more substances may be simultaneously undergo the dissociation. This may prevent the target ion from being identified as a single substance.